In the last thirty or so years, there have been dramatic changes in both sample preparation and testing procedures for numerous applications, including immunoassays, molecular diagnostics, medical microbiology, cell-based assays, and more. Inherent in many of these practical applications are some type of target isolation or separation and some type of target detection method. Improvements in specificity or time-to-results of these tests have come through either improvements in the sample preparation (e.g. isolation or separation of target) or improvements in the detection technologies.
One separation method, namely magnetic separation, has become ubiquitous in the application of isolating targets out of a fluidic sample. These targets range from specific whole cell pathogens, like bacteria, to nonspecific targets, like nucleic acids. A limitation that magnetic separation faces is that the specificity of the separation is governed by the specificity of a single antibody or other bead coating. This limitation stems from the fact that magnetic separation uses a single mechanism (i.e. magnetic forces) to separate the beads with bound target from the rest of the sample. Therefore, it leads to limited specificity and also to a high chance of non-specific binding. This is not only because of the single antibody or bead coating, but because of the sheer number of beads that make it through the process, each of which may have an adherent non-target. The number of magnetic particles in a separation process can be three or four orders of magnitude higher than the number of targets. This can result in non-targets being captured, which can affect methods like cell culturing or PCR, where non-targets can cause false positives or inhibit reactions. In applications like food testing, this limited specificity leads to the need of culturing captured cells on selective or colorimetric growth conditions to indicate a positive identification of the organism. Also in food testing, the presence of inhibiting agents binding to magnetic beads may limit the amount of initial sample that can be used for a PCR reaction.
Furthermore, because performing separation on a single physical mechanism allows for specificity from only a single biomolecule or coating (i.e. a single antibody), existing separation methods are not being used as visual assays. If separation could be used as a visual assay to indicate the presence of a target, there could be improvements in sensitivity and ease of use. However, in order to make the test accurate and practical, this would require the use of double specificity (i.e. like is done with a sandwich immunoassay) during the separation process. This may also require the ability to separate the sample using two different and distinct physical properties (i.e. magnetism in addition to another mechanism).
Lateral flow assays are one of the most common forms of visual assays and they often utilize some form of sandwich immunoassay. However, lateral flow assays also have limitations in both the amount of sample that can be used (for example 100 μL) and the resulting sensitivity of the method. Also, the line that indicates the presence of a target can be difficult to read. Finally, lateral flow assays are one of the few methods that can be performed as a one-step assay; however, they don't necessarily utilize magnetic separation and the advantages that separation provides, including the ability to use larger initial amounts of sample.
Once sample preparation—possibly using magnetic separation—has been completed, many downstream methods exist to detect the presence of the target or if the target is a cell, to measure its growth. These methods may include immunoassays, selective culturing, nucleic-acid techniques, or asynchronous magnetic bead rotation (AMBR). While these detection and culturing methods can all be improved by having better sample separation, there are also improvements that can be made specifically for AMBR.